Particulate hyaluronic acid and flavonoid formulations for cellular delivery of bioactive agents

ABSTRACT

There is presently provided a suspension of immiscible particles in a solution, wherein the particles comprise an agglomeration of a bioactive agent, for example an anti-cancer agent; and a plurality of conjugates of a hyaluronic acid and a flavonoid, for example a catechin-based flavonoid, wherein the particles are on average from about 15 nm to about 300 nm in diameter and wherein the bioactive agent is releasably retained in the particles by the flavonoid. The suspension is useful for delivery of the bioactive agent to cells, including cancer cells. There are also provided a therapeutic formulation comprising the suspension, as well as methods for using the suspension and therapeutic formulation, including for delivery of a bioactive agent to a cell and for treating a disease, including cancer.

CROSS-REFERENCE TO RELATED APPLICATIONS

This patent application is a divisional application of U.S. patentapplication Ser. No. 13/390,097, filed Feb. 10, 2012, entitledPARTICULATE HYALURONIC ACID FORMULATIONS FOR CELLULAR DELIVERY OFBIOACTIVE AGENTS which is a U.S. National Phase application under 35U.S.C. §371 of International Application No. PCT/SG2010/000297, filed onAug. 11, 2010, entitled PARTICULATE HYALURONIC ACID FORMULATIONS FORCELLULAR DELIVERY OF BIOACTIVE AGENTS, which claims benefit of, andpriority from. Singapore patent application No. 200905341-4 filed onAug. 11, 2009, the contents of which have been incorporated byreference.

FIELD OF THE INVENTION

The present invention relates generally to a suspension of immiscibleparticles in a solution, wherein the particles comprise an agglomerationof a bioactive agent and a plurality of conjugates of a hyaluronic acidand a flavonoid; wherein the particles are on average from about 15 nmto about 300 nm in diameter and wherein the bioactive agent isreleasably retained in the particles by the flavonoid.

The present invention also relates to a therapeutic formulationcomprising the suspension and methods for using the suspension andtherapeutic formulation.

BACKGROUND OF THE INVENTION

A main challenge in cancer therapy is the selective delivery ofcytotoxic agents to tumor cells. These cytotoxic agents can be smallmolecules or macromolecules (e.g. proteins, DNA and siRNA), and can haveeither extracellular or intracellular targets. Much research in cancertherapy has focused on improving the targeted delivery of small moleculedrugs such as doxorubicin, which generally have poor selectivity totumor cells.

More recently, the use of monoclonal antibodies such as Herceptin tospecifically target tumor cells that overexpress the cell-surface Her2receptor has emerged as a viable strategy to achieve targeted cancertherapy.^(1,2) Monoclonal antibodies that are used today generallytarget extracellular receptors. However, there is a wide range ofintracellular proteins in cancer cells that can be targeted byantibodies with intracellular targets, otherwise known asintrabodies.^(3,4) An intrabody conjugated to caspase 3 which triggersapoptosis upon antibody-antigen interaction is a prime example of apotential intrabody-based cancer therapy.⁵

Some common methods of delivering intrabodies are: (i) the transfectionof recombinant DNA coding the intrabody into cancer cells, resulting inintracellular expression of the intrabody, and (ii) the fusion ofprotein transduction domains to the intrabody to make it morecell-permeable.⁶ The first method usually requires the use of viralvectors, which raise safety concerns for human clinical use.Furthermore, protein folding and stability of the expressed intrabody inthe successfully transfected cancer cells may be affected by thereducing intracellular environment. In the second method, the intrabodyprotein being delivered is not protected from degradation, and is evenmodified to include a transduction domain, which may compromiseintrabody activity and hence its intracellular function. Neither oftheses existing methods enable the active targeting of tumor cells,which is important to minimize any side effects of the intrabody beingdelivered.

SUMMARY OF INVENTION

In one aspect, there is presently provided a suspension of immiscibleparticles in a solution, wherein the particles comprise an agglomerationof a bioactive agent and a plurality of conjugates of a hyaluronic acidand a flavonoid; wherein the particles are on average from about 15 nmto about 300 nm in diameter and wherein the bioactive agent isreleasably retained in the particles by the flavonoid. In another aspectthere is provided a therapeutic formulation comprising the suspension asdescribe herein. The suspension and therapeutic formulation may be usedto deliver the bioactive agent to cells, including cancer cells.

Thus in one aspect there is provided a suspension of immiscibleparticles in a solution, wherein the particles comprise an agglomerationof a bioactive agent; and a plurality of conjugates of a hyaluronic acidand a flavonoid; wherein the particles are on average from about 15 nmto about 300 nm in diameter and wherein the bioactive agent isreleasably retained in the particles by the flavonoid.

In particular embodiments, the solution is an aqueous solution.

In different particular embodiments, the bioactive agent is releasablyretained in the particles by a hydrophobic bond between the flavonoidand the bioactive agent or by an ionic bond between the flavonoid andthe bioactive agent.

In particular embodiments, the particles are on average from about 50 nmto about 100 nm in diameter.

In certain embodiments, the flavonoid is a catechin-based flavonoid,including for example epigallocatechin gallate.

In particular embodiments, the bioactive agent is an anti-cancer agent.

In different embodiments, the bioactive agent is a protein, includingfor example an intrabody or Granzyme B.

In certain embodiments, the bioactive agent is a bioactive agent that isincapable of entering into a cell when delivered to the cell alone.

In particular embodiments, the HA has a molecular weight of from about5000 to about 10000000 daltons.

In particular embodiments, there is provided the suspension as describedherein wherein the particles are formed by combining in a solution 0.01%w/v to about 1.0% w/v of a bioactive agent and 0.05 μg/ml to about 5000μg/ml of conjugates of a hyaluronic acid and a flavonoid.

In certain embodiments, the particles of the suspension as describedherein further comprise an endosomolytic agent, including for examplepolyethylenimine.

In another aspect, there is provided a therapeutic formulationcomprising the suspension as described herein.

In yet another aspect, there is provided a method for delivery of abioactive agent to a cell, the method comprising contacting the cellwith the suspension or formulation as described herein.

In another aspect, there is provided a method for intracellular deliveryof a bioactive agent to a cell, the method comprising contacting thecell with the suspension or formulation as described herein.

In particular embodiments of the method for delivery of a bioactiveagent to a cell and the method for intracellular delivery of a bioactiveagent to a cell described herein, the cell is a cancer cell.

In different embodiments of the method for delivery of a bioactive agentto a cell and the method for intracellular delivery of a bioactive agentto a cell described herein, the cell is in vitro or in vivo. In oneembodiment, the method comprises administering the particle to a subjectin an amount effective for the treatment of cancer.

In other aspects, there is provided use of the suspension or formulationas described herein for delivery of a bioactive agent to a cell and inthe manufacture of a medicament for delivery of a bioactive agent to acell.

In another aspect there is provided, the suspension or formulation asdescribed herein for use in the delivery of a bioactive agent to a cell.

In other aspects, there is provided use of the suspension or formulationas described herein for intracellular delivery of a bioactive agent to acell and in the manufacture of a medicament for intracellular deliveryof a bioactive agent to a cell.

In another aspect, there is provided the suspension or formulation asdescribed herein for use in the intracellular delivery of a bioactiveagent to a cell.

In particular embodiments of the uses, suspension or formulation asdescribed herein, the cell is a cancer cell.

In one embodiment, the use, suspension or formulation as described is inan amount effective for the treatment of cancer.

In another aspect, there is presently provided a method of formulating asuspension of immiscible particles comprising combining in a solution0.01% w/v to about 1.0% w/v of a bioactive agent; and 0.05 μg/ml toabout 5000 μg/ml of conjugates of a hyaluronic acid and a flavonoid; toform the suspension of particles wherein the particles comprise anagglomeration of the bioactive agent and the conjugates of hyaluronicacid and a flavonoid, wherein the particles are on average from about 15nm to about 300 nm in diameter and wherein the bioactive agent isreleasable retained in the particles by the flavonoid.

In particular embodiments of the present method of formulating asuspension of immiscible particles, the solution is an aqueous solution.

In different particular embodiments, the bioactive agent is releasablyretained in the particles by a hydrophobic bond between the flavonoidand the bioactive agent or by an ionic bond between the flavonoid andthe bioactive agent.

In particular embodiments, the particles are on average from about 50 nmto about 100 nm in diameter.

In certain embodiments of the present method of formulating a suspensionof immiscible particles, the flavonoid is a catechin-based flavonoid,including for example epigallocatechin gallate.

In particular embodiments, the bioactive agent is an anti-cancer agent.

In different embodiments, the bioactive agent is a protein, includingfor example an intrabody or Granzyme B.

In certain embodiments of the present method of formulating a suspensionof immiscible particles, the bioactive agent is a bioactive agent thatis incapable of entering into a cell when delivered to the cell alone.

In particular embodiments, the HA has a molecular weight of from about5000 to about 10000000 daltons.

In certain embodiments, the present method of formulating a suspensionof immiscible particles comprises combining an endosomolytic agent withthe bioactive agent and the conjugates of a hyaluronic acid and aflavonoid to form the suspension and wherein the particles furthercomprise the endosomolytic agent. In particular embodiments, theendosomolytic agent is polyethylenimine.

In particular embodiments, the present method of formulating asuspension of immiscible particles is to formulate a therapeuticformulation.

In another aspect, there is provided a method of treating a diseasecomprising administering to a subject in need thereof an effectiveamount of the suspension or formulation as described herein. In certainembodiments, the disease is cancer.

BRIEF DESCRIPTION OF THE DRAWINGS

In the figures, which illustrate, by way of example only, embodiments ofthe present invention:

FIG. 1. Self-assembly of HA-EGCG and protein into a particle.

FIG. 2. Chemical structure of HA-EGCG conjugates.

FIG. 3. Effect of Lysozyme concentration on particle size ofHA-EGCG/lysozyme particles (□: HA-EGCG 0.75 mg/ml, ▪: HA 0.75 mg/ml, ⋄:HA-EGCG 1 mg/ml, ♦: HA 1 mg/ml).

FIG. 4. Effect of lysozyme concentration on particle homogeneity(measured as sample quality) of HA-EGCG/lysozyme suspension.

FIG. 5. EGCG quenches the intrinsic fluorescence of lysozyme (EX: 280nm) in a concentration-dependent manner.

FIG. 6. EGCG moiety is important for the binding interaction betweenHA-EGCG and lysozyme. HA-EGCG (a) quenches intrinsic lysozymefluorescence in a concentration-dependent manner, and to a larger extentthan HA (b). [lysozyme]=250 μg/ml. Blue: Lysozyme only, Brown:Lysozyme+15 μg/ml HA or HA-EGCG, Green: Lysozyme+125 μg/ml HA orHA-EGCG, Red: Lysozyme+250 μg/ml HA or HA-EGCG.

FIG. 7. Conjugation of EGCG to HA increases the extent to which theintrinsic fluorescence of lysozyme is quenched at a constant EGCGconcentration.

FIG. 8. HA-EGCG decreases the secondary structure content of lysozyme.[lysozyme]=0.5 mg/ml.

FIG. 9. Lysozyme activity. a) inhibition of lysozyme activity at higherconcentrations of HA-EGCG. [lysozyme]=10 μg/ml. Red: HA-EGCG, blue: HA.b) Adding Triton-X to HA-EGCG/lysozyme particles restores lysozymeactivity that is lost during complexation with HA-EGCG. Blue bar (leftbars): lysozyme only, red bars (right bars): HA-EGCG/lysozyme particle.

FIG. 10. Cellular internalization of HA-EGCG a) Rhodamine B-labeledHA-EGCG is internalized by HCT-116 cells much more efficiently thanRhodamine B-labeled HA. b) Lysozyme is internalized more efficiently byHCT-116 cells when delivered with HA-EGCG as a carrier than withoutHA-EGCG. c) Colocalization of HA-EGCG-rhodamine B and FITC-lysozymeshows that the increased lysozyme uptake is due to HA-EGCG acting as acarrier for lysozyme.

FIG. 11. Cell viability of HCT-116 cells incubated with particlescomprising HA-EGCG, Granzyme and PEI.

DETAILED DESCRIPTION

In one aspect, there is provided a suspension of immiscible particles ina solution, wherein the particles comprise an of a bioactive agent; anda plurality of conjugates of a hyaluronic acid and a flavonoid; whereinthe particles are on average from about 15 nm to about 309 nm indiameter and wherein the bioactive agent is releasably retained in theparticles by the flavonoid.

As used herein, suspension refers to a heterogeneous mixture in whichparticles are dispersed throughout a medium, preferably a liquid. Aswould be understood, the particles of a suspension may settle out of themedium but can be re-dispersed, for example through agitation of themixture. As used herein, suspension is not restricted to mixturescomprising particles with a diameter greater than one micrometer butencompasses mixtures comprising particles with diameters of less thanone micrometer including particles with a diameter between 15 and 300nm.

As used herein, agglomeration refers to a mixture or aggregation ofmolecules, moieties or compounds.

The presently described particles may provide delivery of the bioactiveagent contained in the particles to cells. Furthermore, the presentlydescribed particles may provide targeted delivery of the bioactive agentcontained in the particles to cancer cells. The inclusion of hyaluronicacid (HA) in the present particles provides an active targeting sequencefor the CD44 receptor that binds HA and is overexpressed in many cancercell types including colon, breast, ovarian, liver and pancreas cancercells^(7,8) As such, the particles presently provided may specificallytarget cancer cells that overexpress the CD44 receptor for delivery ofthe bioactive agent. In particular embodiments, the bioactive agent isan anti-cancer agent.

Flavonoids, such as (−)-epigallocatechin gallate (EGCG) have the abilityto bind proteins^(11,12) through the formation of non-covalentreversible bonds such as ionic or hydrophobic bonds. Thus, in thepresently described particles, the bioactive agent may be releasablyretained in the particles by interactions between the flavonoid and thebioactive agent including non-covalent bonds such as hydrophobic,hydrogen or ionic bonds. Use of such non-covalent binding to retain thebioactive agent in the particles avoids the need for chemicalconjugation which can irreversibly alter the tertiary structure of thebioactive agent and thus its activity. Furthermore, such non-covalentbonds, while retaining the bioactive agent in the particles when theparticles are in the presently described suspension, may allow forrelease of the bioactive agent from the particles to be easily achievedupon delivery of the bioactive agent to a cell.

Thus, in one embodiment of the present suspension, the bioactive agentis releasably retained in the particles by the flavonoid. In specificembodiments, the bioactive agent is releasably retained in the particlesthrough non-covalent bonds between the bioactive agent and theflavonoid. In different embodiments, the non-covalent bonds may behydrophobic bonds, ionic bonds or hydrogen bonds. In certainembodiments, the non-covalent bonds between the flavonoid and thebioactive agent retain the bioactive agent in the particles when in thepresent suspension but release the bioactive agent from the particlesfollowing delivery of the bioactive agent to a cell.

In particular embodiments, the bioactive may have an affinity for theflavonoid such that the bioactive agent preferentially interacts orbinds with the flavonoid over other compounds, molecules or components.For example, the bioactive agent may have a higher affinity for theflavonoid than for the HA. Thus, in certain embodiments, the bioactiveagent may preferentially, but, releasably interact with the flavonoidsuch that the bioactive agent is preferentially retained in the particle

As used herein, “releasably retained” refers to retainment of thebioactive agent in the present particles in such a manner that thebioactive agent may be subsequently released from the particles. Incertain embodiments, the bioactive agent is released from the particlesby disruption of interactions, including for example non-covalent bonds,between the flavonoid and the bioactive agent.

In certain embodiments, the interaction between the flavonoid and thebioactive agent in the present particles may be characterized bycomplexation between the flavonoid and the bioactive agent such that theflavonoid and the bioactive agent interact to form a complex. As usedherein a “complex” refers to an entity formed from association ofcomponents through specific interactions rather than random associationof the components. As would be understood by a skilled person, a complexmay be comprised of different types of interactions but that theinteractions included within a particular complex are limited to certainspecific types of interactions that are defined by the components orparts of the components that are involved in the interaction as well thetypes of interactions or bonds formed. Thus, in particular embodiments,in the presently described particles the bioactive agent and theplurality of conjugates of a HA and a flavonoid form a complex.

The particles presently provided are formed by the self-assembly of theconjugates of HA and a flavonoid (HA-flavonoid conjugates) and thebioactive agent to form the particles as presently described (FIG. 1).Without being limited to any particular theory, the self-assembly ofHA-flavonoid conjugates and the bioactive agent into the presentlyprovided particles may result from the interactions between theflavonoid and the bioactive agent. In particular embodiments, theself-assembly of HA-flavonoid conjugates and the bioactive agent intothe presently provided particle may result from the formation ofreversible non-covalent binding between the flavonoid and the bioactiveagent.

Without being limited to any particular theory, it appears that incontrast to a polymeric or liposomal carrier, the present particlesprovide a hydrated environment within the particles, which can protectthe bioactive agent from degradation, including degradation by proteasesor the reticulo-endothelial system, hence providing an extendedcirculation time for the bioactive agent. Furthermore, the use ofHA-flavonoid conjugates to form the present particles may reducepotential biocompatibility concerns about delivery system materials asHA has been proven to be a biocompatible material.¹⁰

In particular embodiments, the presently described particles may be ableenter or be transported into a cell and thus the present particles maydeliver the bioactive agent directly inside a cell. Such direct deliveryof the bioactive agent into the cell eliminates the need for viraltransfection and intracellular expression. Furthermore, the presentparticles may provide delivery of the bioactive agent into a cellwithout the need to modify the bioactive agent, for example by adding atransduction domain, in order enable the bioactive agent to traverse theplasma membrane and enter the cell. In particular embodiments, thepresent particles may be used to deliver into a cell a bioactive agentthat ordinarily cannot cross the cell plasma membrane when delivered toa cell on its own.

Thus the present particles may provide delivery of a bioactive agent toa cell and in certain embodiments, targeted delivery of a bioactiveagent to a cancer cell. In particular embodiments, the present particlesmay provide delivery of the bioactive agent directly into the cell. Thepresent particle may provide such delivery without modifications of thebioactive agent to incorporate targeting or transduction domains whichcan alter the functionality or activity of the bioactive agent.

Flavonoids are one of the most numerous and best-studied groups of plantpolyphenols. The flavonoids consist of a large group of low-molecularweight polyphenolic substances naturally occurring in fruits andvegetables, and are an integral part of the human diet. Dried green tealeaves can contain as much as 30% flavonoids by weight, including a highpercentage of flavonoids known as catechins (flavan-3-ol derivatives orcatechin-based flavonoids), including (−)-epicatechin,(−)-epigallocatechin, (+)-catechin, (−)-epicatechin gallate and(−)-epigallocatechin gallate.

In recent years, these green tea catechins have attracted much attentionbecause they have been recognized to have biological and pharmacologicalproperties, including anti-bacterial, anti-neoplastic, anti-thrombotic,vasodilatory, anti-oxidant, anti-mutagenic, anti-carcinogenic,anti-hypercholesterolemic, anti-viral and anti-inflammatory properties,which have been demonstrated in numerous human, animal and in vitrostudies¹⁶⁻¹⁸. These biological and pharmacological properties arepotentially beneficial in preventing diseases and protecting thestability of the genome. Many of the beneficial effects of catechins arethought to be linked to the antioxidant actions of the catechins¹⁹.Among the catechins, (−)-epigallocatechin gallate (EGCG), which is amajor component of green tea, is thought to have the highest activity,possibly due to the trihydroxy B ring and the gallate ester moiety atthe C3 position²⁰⁻²⁴. EGCG has been recognized to have biochemical andpharmaceutical effects including anti-oxidant, anti-carcinogenic, andanti-inflammatory properties²⁵⁻²⁷. EGCG is known to inhibit a vast arrayof biomedically relevant molecular targets and disease-related cellularprocess²⁸ consequently leading to the induction of apoptosis, inhibitionof tumour cell growth, and inhibition of angiogenesis²⁹. Thesebeneficial bioactivities are attributed mostly to the strong bindingability of EGCG to many biological molecules, including peptides andproteins, which affect various enzyme activities and signal transductionpathways³⁰. EGCG is also known as a potent inhibitor of matrixmetalloproteinase (MMP) gclatinases³¹ which play a crucial role intumour metastasis.

Thus, in certain embodiments, if the bioactive agent included in thepresent particles has a therapeutic effect that is also provided by theflavonoid included in the present particles, then the present particlesmay provide therapeutic synergism due to combined delivery of thebioactive agent and the flavonoid to a cell. In one embodiment, thecombination of an anti-cancer agent and a flavonoid, such as EGCG in thepresent particles may provide therapeutic syngergism in the treatment ofcancer.

Formation of HA-Flavonoid Conjugates

The particles presently described are comprised of a bioactive agent,including an anti-cancer agent, and a plurality of conjugates ofhyaluronic acid (HA) and a flavonoid (HA-flavonoid conjugates).

The HA-flavonoid conjugates of the present particles may be comprised ofHA and any suitable flavonoid, as described below.

In different embodiments, the HA is aldehyde-derivatized hyaluronicacid, hyaluronic acid conjugated with aminoacetylaldehyde diethylacetal,or either of the aforementioned hyaluronic acid polymers derivatizedwith tyramine. Methods of synthesizing such HA polymers are known in theart and have been described for example in international application WO2006/124000 and US application 2008/102052, the content of which arefully incorporated herein.

The free aldehyde group on the HA moiety allows for the conjugation ofthe HA in a controlled manner to either the C6 or the C8 position of theA ring, or both, of a flavonoid structure, thus preventing disruption ofthe flavonoid structure, particularly the B and C rings of theflavonoid, and thus preserving the beneficial biological andpharmacological properties of the flavonoid.

The flavonoid may be any flavonoid from the general class of moleculesderived from a core phenylbenzyl pyrone structure, and includesflavones, isoflavones, flavonols, flavanones, flavan-3-ols, catechins,anthocyanidins and chalcones.

In particular embodiments the flavonoid is a catechin or acatechin-based flavonoid. A catechin, or a catechin-based flavonoid isany flavonoid that belongs to the class generally known as catechins (orflavan-3-ol derivatives), and includes catechin and catechinderivatives, including epicatechin, epigallocatechin, catechin,epicatechin gallate and epigallocatechin gallate, and including allpossible stereoisomers of catechins or catechin-based flavonoids. Inparticular embodiments, the catechin-based flavonoid is (+)-catechin or(−)-epigallocatechin gallate. In a particular embodiment, thecatechin-based flavonoid is epigallocatechin gallate (EGCG).

A catechin-based flavonoid to be conjugated to HA may be a singlemonomeric unit of a catechin-based flavonoid or it may be an oligomer ofone or more catechin-based flavonoids. The conjugation of HA to aflavonoid can result in augmentation of the flavonoids biological orpharmacological properties. Furthermore, oligomers of catechin-basedflavonoids tend to have amplified or augmented levels of the biologicaland pharmacological properties associated with catechin-basedflavonoids, and may even have reduced pro-oxidant effects that aresometimes associated with monomeric catechin-based flavonoids. Thus inone embodiment, an oligomerized catechin-based flavonoid havingamplified or augmented flavonoid properties is conjugated to HA.

Oligomers of catechin-based flavonoids that can be conjugated to HA,such as polymers, are known, and include oligomers prepared throughenzyme-catalyzed oxidative coupling and through aldehyde-mediatedoligomerization, for example as described in published internationalapplication WO 2006/124000 and published US application 2008/102052, thecontents of which are fully incorporated by reference herein.

An aldehyde-mediated oligomerization process results in an unbranchedoligomer that has defined linkages, for example through carbon-carbonlinkages such as CH—CH₃ bridges linked from the C6 or C8 position on theA ring of one monomer to the C6 or C8 position on the A ring of the nextmonomer, including in either possible stereoconfiguration, whereapplicable. Thus, the CH—CH₃ linkage may be between the C6 position ofthe A ring of one monomer and either of the C6 or C8 position of thenext monomer or it may be between the C8 position of the A ring of thefirst monomer and either of the C6 or C8 position of the next monomer.

The oligomer of catechin-based flavonoid to be conjugated to HA, forexample a polymer, may be of 2 or more monomeric units linked together.In certain embodiments, the catechin-based flavonoid oligomer has from 2to 100 monomer units, from 10 to 100, from 2 to 80, from 10 to 80, from2 to 50, from 10 to 50, from 2 to 30, from 10 to 30, from 20 to 100,from 30 to 100 or from 50 to 100 monomeric units.

HA may be conjugated to the flavonoid by any suitable means known in theart that provides attachment of HA to the flavonoid to form a conjugatecapable of being formed into particles as described herein withoutdisruption of the polyphenol structure of the flavonoid.

In one embodiment, HA may be conjugated to a flavonoid by “aldehydemediated conjugation” wherein HA is reacted with the flavonoid in thepresence of an acid catalyst, the HA moiety having a free aldehydegroup, or a group that is able to be converted to a free aldehyde groupin the presence of acid. Aldehyde-mediated conjugation of HA to aflavonoid can result in attachment of HA at the C6 and/or C8 position ofthe flavonoid A ring, which does not disrupt or affect the B and C ringsof the flavonoid or the various hydroxyl groups on the flavonoid.Formation of HA-flavonoid conjugates by aldehyde mediated conjugation isdescribed in published international application WO 2006/124000 andpublished US application 2008/102052, the contents of which are fullyincorporated be reference herein.

In other embodiments, the flavonoid to be conjugated to the HA may bemodified to form a flavonoid derivative that comprises a functionalgroup that is suitable for conjugation with the HA. In particularembodiments, the flavonoid is modified to form a flavonoid derivativethat comprises a terminal group that is suitable for conjugation withthe HA. In different embodiments, the flavonoid may be modified tocomprise a carboxyl, an amine or a succinimide functional group. Thus inparticular embodiments, the HA-flavonoid conjugate may be formed byfirst preparing a flavonoid derivative comprising a functional group,for example a terminal group, suitable for conjugation with HA. Thisflavonoid derivative is then reacted with HA to form the HA-flavonoidconjugate.

In a particular embodiment, the HA-flavonoid conjugate is comprised ofHA conjugated to a catechin-based flavonoid and the conjugation iscarried out by aldehyde mediated conjugation as defined above. Thus, theconjugation reaction may involve conjugation of a HA moiety containing afree aldehyde group or a group that is able to be converted to a freealdehyde group in the presence of acid to a catechin-based flavonoid.Thus in one embodiment, the HA-flavonoid conjugate may be synthesizedusing acid catalysis of a condensation of the aldehyde group of HA witha catechin-based flavonoid, or using acid to convert a functional groupon HA to a free aldehyde prior to condensation of the aldehyde groupwith the catechin-based flavonoid. In a particular embodiment, thecatechin-based flavonoid is EGCG.

To conjugate HA and the flavonoid, the HA moiety and the flavonoid maybe separately dissolved in a suitable solvent. For example, HA with thefree aldehyde may be added by dropwise addition, to a solutioncontaining the flavonoid, in the presence of an acid, for example at apH from about 1 to about 5, or for example at pH of about 1. Thereaction is allowed to go to completion. Following the conjugationreaction, excess unreacted flavonoid can be removed from the conjugatedcomposition, for example by dialysis or by molecular sieving.

In another embodiment, the HA moiety may be dissolved in deionized ordistilled water and mixed with a solution comprising the flavonoiddissolved in dimethyl sulfoxide (DMSO). The pH of the solution isadjusted to about 1 by addition of an acid, for example HCl and thereaction is allowed to go to completion, for example by stirring at roomtemperature for about 24 hours. Following the conjugation reaction, theconjugate may be purified from the solution, for example by dialysis.

The ratio of flavonoid to HA, may be varied, so that there is only oneHA moiety attached to the flavonoid, or so that there is a flavonoidattached at more than one position on the HA moiety or so that theflavonoid has two HA moieties attached, for example one at either of theC6 and C8 positions of a catechin-based flavonoid.

The ratio of HA moiety to flavonoid in the conjugate can be controlledthrough the ratio of starting reagents. For example, when the molarratio of HA to flavonoid is about 1, a single HA moiety will be attachedto a single flavonoid moiety (either monomeric or oligomeric may beused). However, at higher concentrations of HA, for example at a 10:1molar ratio of HA to flavonoid, a composition having a tri-blockstructure of HA-flavonoid-HA may be obtained.

Similarly, the degree of conjugation of HA with the flavonoid can bevaried by varying the concentrations of HA and flavonoid in theconjugation reaction. The “degree of conjugation” as used herein refersto the number of flavonoid molecules per 100 units of HA. For example, a50% degree of conjugation means that there are 50 flavonoid moleculesper 100 units of HA.

Since HA has multiple sites that may react with a flavonoid during theconjugation reaction, by varying the concentration of the flavonoid inthe starting reaction, it is possible to vary the degree of conjugationbetween HA and the flavonoid.

The ratio of HA to flavonoid in the starting reagents for forming theHA-flavonoid conjugates may be varied to adjust the degree ofconjugation of the HA with the flavonoid in the resulting HA-flavonoidconjugates and thus the ratio of HA to flavonoid present in a particleformed from these HA-flavonoid conjugates.

In a particular embodiment, the HA-flavonoid conjugate is a conjugate ofHA and EGCG and the conjugate is synthesized in a two-step procedure. Inthe first step protected aldehyde groups are introduced to HA byconjugating diethoxyethyl amine (DA) to HA though NHS/EDC chemistry forform HA-DA conjugates. The HA-EGCG conjugate is then formed bydeprotection of the HA-DA conjugates at a pH of 1 to allow conjugationof EGCG to the aldehyde groups.

In different embodiments, the HA moiety has a molecular weight of fromabout 5000 to about 10,000,000 daltons, at least about 5000 daltons, atleast about 10,000 daltons, at least about 20,000 daltons, at leastabout 30,000 daltons, at least about 40,000 daltons, at least about50,000 daltons, at least about 60,000 daltons, at least about 70,000daltons, at least about 80,000 daltons, at least about 90,000 daltons,at least about 100,000 daltons, at least about 150,000 daltons, at leastabout 200,000 daltons, at least about 250,000 daltons, at least about300,000 daltons, at least about 350,000 daltons, at least about 400,000daltons, at least about 450,000 daltons, at least about 500,000 daltons,at least about 550,000 daltons, at least about 600,000 daltons, at leastabout 650,000 daltons, at least about 700,000 daltons at least about750,000 daltons, at least about 800,000 daltons, at least about 850,000daltons, at least about 900,000 daltons, at least about 950,000 daltons,at least about 1,000,000 daltons, at least about 1,500,000 daltons, atleast about 2,000,000 daltons, at least about 2,500,000 daltons, atleast about 3,000,000 daltons, at least about 3,500,000 daltons, atleast about 4,000,000 daltons, at least about 4,500,000 daltons, at,least about 5,000,000 daltons, at least about 5,500,000 daltons, atleast about 6,000,000 daltons, at least about 6,500,000 daltons, atleast about 7,000,000 daltons, at least about 7,500,000 daltons, atleast about 8,000,000 daltons, at least about 8,500,000 daltons, atleast about 9,000,000 daltons, at least about 9,500,000 daltons, or atleast about 10,000,000 daltons.

Formation of Particles Comprising HA-Flavonoid Conjugates and aBioactive Agent

In one aspect, there is presently provided a suspension of immiscibleparticles in an solution, wherein the particles comprise anagglomeration of a bioactive agent; and a plurality of conjugates of ahyaluronic acid and a flavonoid; wherein the particles are on averagefrom about 15 nm to about 300 nm in diameter and wherein the bioactiveagent is releasable retained in the particles by the flavonoid.

The solution may be any solution that is suitable for dispersion of thepresently described particles to form the present suspension. Thesolution is preferably non-toxic and suitable for pharmacological use.In one embodiment, the solution is an aqueous solution.

The bioactive agent may be any agent that has a biological,pharmacological or therapeutic effect in a body or cell, and includeswithout limitation a protein, a nucleic acid, a small molecule or adrug. A bioactive agent that is a protein may be for example a peptide,an antibody, an intrabody, a hormone, an enzyme, a growth factor or acytokine. A bioactive agent that is a nucleic acid may be for examplesingle stranded or double stranded DNA or RNA, a short hairpin RNA, ansiRNA, or may comprise a gene encoding a therapeutic product. Alsoincluded in the scope of bioactive agent are antibiotics,chemotherapeutic agents, antihypertensive agents, anti-cancer agents,anti-bacterial agents, anti-neoplastic agents, anti-thrombotic agents,vasodilatory agents, anti-oxidants, anti-mutagenic agents,anti-carcinogenic agents, anti-hypercholesterolemic agents, anti-viralagents and anti-inflammatory agents.

In particular embodiments, the bioactive agent delivered into the cellmay be an agent that is unable to traverse the plasma membrane and enterthe cell when delivered to the cell on its own. For example, some agentsmay be unable to traverse the plasma membrane on their own due to theirsize, hydrophobicity, hydrophilicity or charge.

In particular embodiments, the bioactive agent may be an anti-canceragent. As used herein, “anti-cancer agent” refers to any agent that hasa biological, pharmacological or therapeutic effect in a body for thetreatment of cancer or that has an anti-cancer effect on a cell,including an anti-tumour effect, such as a cytotoxic, apoptotic,anti-mitotic anti-angiogenesis or inhibition of metastasis effect.“Anti-cancer effect” as used herein is intended to include inhibition orreduction of tumour cell growth, inhibition or reduction ofcarcinogenesis, killing of tumour cells, or inhibition or reduction ofcarcinogenic or tumourogenic properties of a cell, including a tumourcell.

In particular embodiments, the anti-cancer agent may be for example apeptide, an antibody, an intrabody, an enzyme or a cytotoxic protein. Inparticular embodiments, the anti-cancer agent is an intrabody orGranzyme B. Also included in the scope of an anti-cancer agent arechemotherapeutic agents, anti-cancer agents, anti-neoplastic agents,anti-oxidants, anti-mutagenic agents and anti-carcinogenic agents.

The anti-cancer agent may be, for example, herceptin, TNP470,tastuzumab, bevacizumab, rituximab, erlotinib, daunorubicin,doxorubicin, etoposide, vinblastine, vincristine, pacitaxel,methotrexate, 5-fluorouracil, gemcitabine, arabinosylcytosine,altretamine, asparaginase, bleomycin, capecitabine, carboplatin,carmustine, BCNU, cladribine, cisplatin, cyclophosphamide, cytarabine,dacarbazine, dactinomycin, actinomycin D, docetaxel, doxorubicin,doxorubicin, imatinib, doxorubicin liposomal, VP-16, fludarabine,gemcitabine, hydroxyurea, idarubicin, ifosfamide, irinotecan, CPT-11,methotrexate, mitomycin, mitotane, mitoxantrone, topotecan, vinblastine,vincristine, vinorelbine or an antibody for use in immunotherapy.

References herein to embodiments that include a bioactive agent aremeant to exemplify, in addition, embodiments in which an agent, such asa non-bioactive agent, is substituted for the bioactive agent. Thus, inparticular embodiments there is presently provided a suspension ofimmiscible particles in a solution, wherein the particles comprise anagglomeration of an agent; and a plurality of conjugates of a hyaluronicacid and a flavonoid; wherein the particles are on average from about 15nm to about 300 nm in diameter and wherein the agent is releasablyretained in the particles by the flavonoid. As used herein “agent” maybe a bioactive agent or a non-bioactive agent. As used herein “anon-bioactive agent” refers to an agent that does not have a biological,pharmacological or therapeutic effect in a body or cell and includeswithout limitation a protein, a nucleic acid or a small molecule thatdoes not have a biological, pharmacological or therapeutic effect in abody or cell. Thus, in one embodiment the presently provided particlesmay comprise an agent that is a non-bioactive agent. In a particularembodiment, the agent is an inert compound, such as an inert marker foridentifying particular cells in a cell population.

As described above, the present particles are formed by theself-assembly of a plurality of HA-flavonoid conjugates and thebioactive agent to form the particles as presently described.

To form the particles presently described, the HA-flavonoid conjugatesare mixed with the bioactive agent in suitable reaction conditions andat suitable concentrations of the HA-flavonoid conjugates and bioactiveagent to self-assemble to form the presently described particles thatare from about 15 nm to about 300 nm.

The formation of the present particles is dependent on both theconcentration of HA-flavonoid conjugates and the concentration of thebioactive agent which both may effect the self-assembly of theHA-flavonoid conjugates and the bioactive agent and the size of theparticles formed.

To form the present particles, the HA-flavonoid conjugates are generallyprovided at concentrations an order of magnitude less then theconcentrations that result in formation of the HA-flavonoid conjugatesinto a hydrogel. In different embodiments, the concentration ofHA-flavonoid conjugates may be from about 0.05 μg/ml to about 5000μg/ml, from about 10 μg/ml to about 5000 μg/ml, from about 10 μg/ml toabout 1000 μg/ml at least about 0.05 μg/ml, at least about 0.1 μg/ml, atleast about 0.5 μg/ml, at least about 1 μg/ml, at least about 5 μg/ml,at least about 10 μg/ml, at least about 20 μg/ml, at least about 30μg/ml, at least about 40 μg/ml, at least about 50 μg/ml, at least about60 μg/ml, at least about 70 μg/ml, at least about 80 μg/ml, at leastabout 90 μg/ml, at least about 100 μg/ml, at least about 125 μg/ml, atleast about 150 μg/ml, at least about 175 μg/ml, at least about 200μg/ml, at least about 225 μg/ml, at least about 250 μg/ml, at leastabout 275 μg/ml, at least about 300 μg/ml at least about 325 μg/ml, atleast about 350 μg/ml, at least about 375 μg/ml, at least about 400μg/ml, at least about 425 μg/ml, at least about 450 μg/ml, at leastabout 475 μg/ml, at least about 500 μg/ml, at least about 525 μg/ml, atleast about 550 μg/ml, at least about 575 μg/ml, at least about 600μg/ml, at least about 625 μg/ml, at least about 650 μg/ml, at leastabout 675 μg/ml, at least about 700 μg/ml, at least about 725 μg/ml, atleast about 750 μg/ml, at least about 800 μg/ml, at least about 825μg/ml, at least about 850 μg/ml, at least about 875 μg/ml, at leastabout 900 μg/ml, at least about 925 μg/ml, at least about 950 μg/ml, atleast about 975 μg/ml, at least about 1000 μg/ml, at least about 1500μg/ml, at least about 2000 μg/ml, at least about 2500 μg/ml, at leastabout 3000 μg/ml, at least about 3500 μg/ml, at least about 4000 μg/ml,at least about 4500 μg/ml or at least about 5000 μg/ml.

As discussed above, the formation of the present particles is alsodependant on the concentration of the bioactive agent which can affectthe self-assembly and size of the particles described herein. Increasingthe concentration of the bioactive agent may increase or decrease thesize of the particles formed. The effect of increasing the concentrationof the bioactive agent may depend on the type of bioactive agent and theamount of bioactive agent used. For example, as demonstrated in FIG. 3of the present application, at lower amounts of the bioactive agent anincrease in the concentration of the bioactive agent can result in areduction in the size of the particles formed whereas at higher amountsof the bioactive agent an increase in the concentration of the bioactiveagent can result in an increase in the size of the particles formed.

In different embodiments, the concentration of the bioactive agent maybe from about 0.01% w/v to about 1.0% w/v from about 0.1% w/v to about0.75% w/v, at least about 0.01% w/v, at least about 0.02% w/v, at leastabout 0.03% w/v, at least about 0.04% w/v, at least about 0.05% w/v, atleast about 0.06% w/v, at least about 0.07% w/v, at least about 0.08%w/v, at least about 0.09% w/v, at least about 0.1% w/v, at least about0.15% w/v, at least about 0.2% w/v, at least about 0.25% w/v, at leastabout 0.3% w/v, at least about 0.35% w/v, at least about 0.4% w/v, atleast about 0.45% w/v, at least about 0.5% w/v, at least about 0.55%w/v, at least about 0.6% w/v, at least about 0.65% w/v, at least about0.7% w/v, at least about 0.75% w/v, at least about 0.8% w/v, at leastabout 0.85% w/v, at least about 0.90% w/v, at least about 0.95% w/v orat least about 1.0% w/v.

As would be understood by a skilled person, the concentration of thebioactive agent and the HA-flavonoid conjugates required to form theappropriate size particles to form the particles presently described maydiffer depending on the type of HA-flavonoid conjugates and type ofbioactive agent. Furthermore, a skilled person would appreciate that therequired concentrations of the bioactive agent and the HA-flavonoidconjugate to form the present particles may be relative to each othersuch that the required concentration of the bioactive agent may dependon the concentration of the HA-flavonoid conjugates and vice versa.

A person skilled in the art can use known methods and techniques todetermine, based on the above factors, the relative concentrations ofthe bioactive agent and the HA-flavonoid conjugates required to form theappropriate size particles to form the particles presently described andthus the suspension and therapeutic formulation presently described.

Thus in one aspect, there is presently provided a method of formulatinga suspension of immiscible particles comprising combining in a solution0.01% w/v to about 1.0% w/v of a bioactive agent and 0.05 μg/ml to about5000 μg/ml of conjugates of a hyaluronic acid and a flavonoid to formthe suspension of particles wherein the particles comprise anagglomeration of the bioactive agent and the conjugates of hyaluronicacid and a flavonoid, wherein the particles are on average from about 15nm to about 300 nm in diameter and wherein the bioactive agent isreleasably retained in the particles by the flavonoid.

In contrast to larger structures, such as hydrogels, the size of theparticles presently provided may allow for the particles to enter intocells, including cancer cells. Furthermore, the size of the presentparticles may provide increased mobility of the particles, as comparedto larger structures, throughout the cell culture, body or extracellularenvironment that allows the particles to circulate and move betweencells and towards target cancer cells.

As used herein “diameter” refers to hydrodynamic diameter as measured bydynamic light scattering. As would be understood by a skilled person, a“hydrodynamic diameter” refers to how a particle diffuses within afluid. The diameter obtained by dynamic light scattering is that of asphere that has the same translational diffusion coefficient as theparticle being measured. In certain embodiments, the particles“diameter” may refer to or be referred to as the particles “apparentdiameter” as the measured diameter may be a calculated average diameterbased on one or more measurements of the physical properties of theparticles.

In different embodiments, the present particles are on average fromabout 15 nm to about 300 nm, from about 50 to about 100 nm, at leastabout 15 nm, at least about 20 nm, at least about 25 nm, at least about30 nm, at least about 35 nm, at least about 40 nm, at least about 45 nm,at least about 50 nm, at least about 55 nm, at least about 60 nm, atleast about 65 nm, at least about 70 nm, at least about 75 nm, at leastabout 80 nm, at least about 85 nm, at least about 90 nm, at least about95 nm, at least about 100 nm, at least about 125 nm, at least about 150nm, at least about 175 nm, at least about 200 nm, at least about 225 nm,at least about 250 nm, at least about 275 nm or at least about 300 nm indiameter.

As discussed above, the present particles may be used to deliver thebioactive agent to a cell. In certain embodiments, the present particlesmay deliver the bioactive agent directly into a cell. In order for theparticles to deliver the bioactive agent to a cell, the particles shouldbe suitably sized to permit the particle to move through the cellculture, body or extracellular environment. In addition, if thebioactive agent is to be delivered into the cell, the particles shouldbe suitably sized to permit entry of the particle into the cell. Thus,in particular embodiments, the present particles have an averagediameter that permits the particles to move through a cell culture, bodyor extracellular environment or to enter into a cell.

A skilled person would be able to readily determine the size andstability of a particle using methods and techniques known in the art,including Dynamic Light Scattering as disclosed, in the Examples below.

The present particles are immiscible. As used herein “immiscible” refersto particles that do not aggregate with other particles or additionalHA-flavonoid conjugates or bioactive agents to form large particles orlarge aggregates substantially greater in size then the particlespresently described. As would be understood by a skilled person,intermediate particles of less then about 300 nm may aggregate togetherin order to form the present particles. Such intermediate particles maybe of different sizes. However, the present particles will not aggregatewith each other to form particles that are on average greater than about300 nm in diameter.

The present particles are stable in a solution. In particularembodiments, the present particles are stable in an aqueous solution. Asused herein, a “stable” particle refers to a particle that remainsimmiscible and that does not disintegrate or dissolve in a solution, forexample an aqueous solution, for a sufficient length of time for theintended use of the particles. For example, in one embodiment, thepresent particles may remain immiscible and will not disintegrate ordissolve in an aqueous solution for a sufficient amount of time topermit use of the presently described suspension or therapeutic agentfor delivery of the bioactive agent to a cell. In another embodiment,the present particles may remain immiscible and will not disintegrate ordissolve in aqueous solution for a sufficient amount of time to permitstorage of the presently described suspension or therapeutic formulationfor a desired amount of time.

In different embodiments, the present particles are stable in a solutionfrom at least about 1 hour to at least about 12 weeks, from at leastabout 1 day to at least about 7 days, from at least about 1 hour to atleast about 24 hours, at least about 1 hour, at least about 2 hours, atleast about 3 hours, at least about 4 hours, at least about 5 hours, atleast about 6 hours, at least about 7 hours, at least about 8 hours, atleast about 9 hours, at least about 10 hours, at least about 11 hours,at least about 12 hours, at least about 13 hours, at least about 14hours, at least about 15 hours, at least about 16 hours, at least about17 hours, at least about 18 hours, at least about 19 hours, at leastabout 20 hours, at least about 21 hours, at least about 22 hours, atleast about 23 hours, at least about 24 hours, at least about 1 day, atleast about 2 days, at least about, 3 days, at least about 4 day, atleast about 5 days, at least about 6 days, at least about 7 days, atleast about 1 week, at least about 2 weeks, at least about 3 weeks, atleast about 4 weeks, at least about 5 weeks, at least about 6 weeks, atleast about 7 weeks, at least about 8 weeks, at least about 9 weeks, atleast about 10 weeks, at least about 11 weeks or at least about 12weeks.

In a particular embodiment, the present particles further comprise anendosomolytic agent. Thus, in one embodiment, the present method offormulating a suspension of immiscible particles comprises combining anendosomolytic agent with the bioactive agent and the conjugates of ahyaluronic acid and a flavonoid to form the suspension and wherein theparticles further comprise the endosomolytic agent.

As used herein “endosomolytic agent” refers to any agent that canmediate the release of particles from an endosome into the cytosol of acell. For example, in particular embodiments, the endosomolytic agentmay be polyethylenimine, melittin, an endosomolytic peptide or anendosomolytic protein.

In particular embodiments, the present particle may be transported intoa cell through endocytosis. Without being limited to any particulartheory, the transport of the present particle into a cell throughendocytosis may be mediated by the binding of HA in the present particleto the CD44 receptor on the cell surface.

In endocytosis, particles are transported into the cell byinternalization in endosomes which transport the particles from the cellsurface into the cytosol. An endosomolytic agent may facilitate therelease of the particles from the endosome into the cytosol by lysingthe endosome.

As would be understood, when the bioactive agent of the presentparticles is an endosomolytic agent, for example an endosomolyticpeptide or endosomolytic protein, it may not be necessary to include anadditional endosomolytic agent.

The endosomolytic agent contained in the present particle is preferablynon-toxic and suitable for pharmacological use. In particularembodiments, the activity of the endosomolytic agent is pH-sensitive andthe endosomolytic agent is activated upon exposure to the low pH of theendosome.

In particular embodiments, the endosomolytic agent is polyethylenimine.

In a particular embodiment, there is presently provided a suspension ofimmiscible particles in an aqueous solution, wherein the particlescomprise an agglomeration of a bioactive agent; and a plurality ofconjugates of a hyaluronic acid and EGCG; wherein the particles are onaverage from about 15 nm to about 300 nm in diameter and wherein thebioactive agent is releasably retained in the particles by the EGCG as aresult of the non-covalent bonds between the EGCG and the bioactiveagent. In certain embodiments, the bioactive agent is releasablyretained in the particles by hydrophobic bonds between the EGCG and thebioactive agent. In other embodiments, the bioactive agent is releasablyretained in the particles by ionic bonds between the EGCG and thebioactive agent.

In another particular embodiment, there is presently provided asuspension of immiscible particles in an aqueous solution, wherein theparticles comprise an agglomeration of a bioactive agent; and aplurality of conjugates of a hyaluronic acid and EGCG and anendosomolytic agent; wherein the particles are on average from about 15nm to about 300 nm in diameter and wherein the bioactive agent isreleasably retained in the particles by the EGCG. In certainembodiments, the endosomolytic agent is polyethylenimine. In certainembodiments, the bioactive agent is releasably retained in the particlesby hydrophobic bonds between the EGCG and the bioactive agent. In otherembodiments, the bioactive agent is releasably retained in the particlesby ionic bonds between the EGCG and the bioactive agent.

Methods of Use

The suspension described herein may be used to deliver a bioactiveagent, including an anti-cancer agent, to a cell, including a cancercell. The HA of the present particles can bind to the CD44 receptorwhich is overexpressed in many cell types and thus the present particlesmay specifically target cancer cells for the delivery of the bioactiveagent.

Thus in one aspect, there is provided a method for delivery of abioactive agent to a cell, the method comprising contacting the cellwith the suspension or formulation as described herein.

In certain embodiments, the present particle may deliver the bioactiveagent directly into the cell. Thus in another aspect, there is presentlyprovided a method for intracellular delivery of a bioactive agent to acell, the method comprising contacting the cell with the suspension orformulation as described herein.

In particular embodiments, the bioactive agent delivered into the cellmay be an agent that ordinarily is unable to traverse the plasmamembrane and enter the cell on its own.

In certain embodiments, the cell is a cancer cell. As discussed above,the present particles may selectively target cancer cells for deliveryof the bioactive agent.

As used herein, “delivering” a bioactive agent to a cell refers toproviding the agent in sufficiently close proximity to the cell suchthat the agent can exert its biological effects on the cell.

As used herein, “contacting” a cell refers to providing or administeringthe suspension in a manner that enables the presently describedparticles to deliver the bioactive agent to the cell. In vitro, forexample, contacting the cell may comprise adding the suspension to thecell culture media. In vivo, for example, contacting the cell maycomprise administering the suspension to a subject as a pharmaceuticalcomposition.

Following delivery of the bioactive agent to the cell, the bioactiveagent may be released from the present particles to exert its biologicaleffects. Without being limited to any particular theory, the bioactiveagent may be released from the present particles by disassociation ofthe particles as a result of the disruption of non-covalent reversiblebonds between the bioactive agent and the flavonoid. In differentembodiments this disruption may be achieved outside or inside the cell.In particular embodiments, the particle may be disassociated throughinteraction with molecules of the plasma membrane, interaction withintracellular molecules, through the addition of a disassociation agent,for example. Triton-X, or through changes in pH.

In particular embodiments, the bioactive active agent is an anti-canceragent and the present method provides for delivery of an anti-canceragent to a cancer cell. In certain embodiments, there is provideddelivery of the anti-cancer agent directly into the cell.

In particular embodiments, the cell may be a cell located in a subjectin need of treatment for a disease or disorder. For example, the cellmay be a cell within a subject having cancer, a subject requiringtreatment for cancer or a subject in which prevention of cancer isdesired. In some embodiments, the subject is a human subject.

The term “cell” as used herein includes a single cell, a plurality ofcells or a population of cells where context permits, unless otherwisespecified. The cell may be an in vitro cell, including a cell explantedfrom a subject. The cell may be a cell grown in batch culture or intissue culture plates. Alternatively, the cell may be an in vivo cell ina subject. In some embodiments, the subject is a human subject. Incertain embodiments, the subject is a subject in need of treatment for adisease or disorder. Similarly, reference to “cells” also includesreference to a single cell where context permits, unless otherwisespecified.

As used herein, “cancer cell” refers to any cancer cell thatover-expresses the CD44 receptor. A cancer cell refers to a cell thatexhibits abnormal cell growth, reduced or loss of control over celldivision and the potential to invade nearby tissues. Some cancer cellsmay display metastasis in which the cell spreads to other locations inthe body. Some cancer cells may form tumours. Cancer cells may include,for example, sarcoma, carcinoma, lymphoma or blastoma cells.

“Cancer” as used herein encompasses a class of diseases in which cellsexhibit abnormal cell growth and the potential to invade nearby tissues.In some forms of cancer, the abnormal cells may also spread to otherlocations in the body. Different types of cancer include for example,breast cancer, colorectal cancer, brain cancer, prostate cancer,cervical cancer, ovarian cancer, bone cancer, skin cancer, lung cancer,pancreatic cancer, bladder cancer, gallbladder cancer, kidney cancer,esophageal cancer, Hodgkin lymphoma, non-Hodgkin lymphoma, laryngealcancer, leukemia, multiple myeloma, oral cancer, pleural mesothelioma,small intestine cancer, testicular cancer, uterine cancer, thyroidcancer and stomach cancer.

The term “treatment” refers to an approach for obtaining beneficial ordesired results, including clinical results. Beneficial or desiredclinical results can include, but are not limited to, alleviation oramelioration of one or more symptoms or conditions, diminishment ofextent of disorder or disease, stabilization of the state of disease,prevention of development of disorder or disease, prevention of spreadof disorder or disease, delay or slowing of disorder or diseaseprogression, delay or slowing of disorder or disease onset, ameliorationor palliation of the disorder or disease state, and remission, whetherpartial or total. “Treatment” can also mean prolonging survival of asubject beyond that expected in the absence of treatment. “Treatment”can also mean inhibiting the progression of disorder or disease, slowingthe progression of disorder or disease temporarily, although in someinstances, it involves halting the progression of the disorder ordisease permanently.

The term “effective, amount” or “an amount effective” as used hereinmeans an amount effective at dosages and for periods of time necessaryto achieve a desired result. For example, the panicle may beadministered in quantities and dosages necessary to deliver a bioactiveagent which may function to alleviate, improve, mitigate, ameliorate,stabilize, prevent the spread of, slow or delay the progression of orcure a disease or disorder, or to inhibit, reduce or impair the activityof a disease-related enzyme. A disease-related enzyme is an enzymeinvolved in a metabolic or biochemical pathway, which when the pathwayis interrupted, or when regulatory control of the enzyme or pathway isinterrupted or inhibited, the activity of the enzyme is involved in theonset or progression of a disease or disorder. In particularembodiments, the particle may be administered in quantities and dosagesnecessary to deliver an anti-cancer agent for the treatment of cancer.

The effective amount of the suspension to be administered to a subjectcan vary depending on many factors such as the pharmacodynamicproperties of the suspension or the particles in the suspension,including the properties of the HA-flavonoid conjugates and thebioactive agent in the particles, the mode of administration, the age,health and weight of the subject, the nature and extent of the disorderor disease state, the frequency of the treatment and the type ofconcurrent treatment, if any. Furthermore, the effective amount may varydepending on the concentration of the bioactive agent provided in theparticles.

One of skill in the art can determine the appropriate amount based onthe above factors. The suspension may be administered initially in asuitable amount that may be adjusted as required, depending on theclinical response of the subject. The effective amount of the presentsuspension can be determined empirically and depends on the maximalamount of the suspension that can be administered safely. However, theamount of suspension administered is preferably the minimal amount thatproduces the desired result.

Therefore, there is provided a pharmaceutical composition comprising thesuspension as described herein. The pharmaceutical composition mayfurther include a pharmaceutically acceptable diluent or carrier. Thepharmaceutical composition may routinely contain pharmaceuticallyacceptable concentration of salts, buffering agents, preservatives andvarious compatible carriers. For all forms of delivery, the particle maybe formulated in a physiological salt solution.

The proportion and identity of the pharmaceutically acceptable diluentor carrier is determined by the chosen route of administration,compatibility with biologically active proteins if appropriate, andstandard pharmaceutical practice.

The pharmaceutical composition can be prepared by known methods for thepreparation of pharmaceutically acceptable compositions suitable foradministration to subjects, such that an effective amount of theparticle and any additional active substance or substances is combinedin a mixture with a pharmaceutically acceptable vehicle. Suitablevehicles are described, for example, in Remington's PharmaceuticalSciences (Remington's Pharmaceutical Sciences, Mack Publishing Company,Easton, Pa., USA 1985). On this basis, the compositions may include theparticle in association with one or more pharmaceutically acceptablevehicles or diluents, and contained in buffer solutions with a suitablepH and iso-osmotic with physiological fluids.

Under ordinary conditions of storage and use, such pharmaceuticalcompositions may contain a preservative to prevent the growth ofmicroorganisms, and that will maintain any biological activity of theparticle and the bioactive agent. A person skilled in the art would knowhow to prepare suitable formulations. Conventional procedures andingredients for the selection and preparation of suitable formulationsare described, for example, in Remington's Pharmaceutical Sciences andin The United States Pharmacopeia: The National Formulary (USP 24 NF19)published in 1999. Alternatively, the flavonoid conjugate hydrogel maybe formulated at a time sufficiently close to use by mixing thecomponents, without the need for preservatives.

Thus in one aspect, there is provided, a therapeutic formulationcomprising the suspension as described herein. In another aspect, thereis provided a method of treating a disease comprising administering to asubject in need thereof an effective amount of the suspension orformulation as described herein.

Uses of the suspension and formulation describe herein for delivery of abioactive agent to a cell and in the preparation of a medicament fordelivery of a bioactive agent to a cell are also contemplated.

Uses of the suspension and formulation describe herein for intracellulardelivery of a bioactive agent to a cell and in the preparation of amedicament for intracellular delivery of a bioactive agent to a cell arealso contemplated.

The present methods and compounds are further exemplified by way of thefollowing non-limiting examples.

EXAMPLES Materials

Sodium hyaluronate (HA) (MW=90 KDa, density=1.05 g/cm³) was kindlyprovided by Chisso Corporation (Tokyo, Japan). Diethoxyethyl amine (DA),N-hydroxysuccinimide (NHS),1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC.HCl),lysozyme from chicken egg white and Micrococcus lysodeikticus were allpurchased from Sigma-Aldrich. Lissamine rhodamine B ethyldiamine andfluorescein isothiocynate (FITC) were purchased from Invitrogen.Phosphate buffer saline (PBS, 150 mM, pH 7.3) was supplied by mediapreparation facility in Biopolis, Singapore.

ExGen 500 Transfection Reagent (Linear poly(ethyleneimine) (PEI) 22 kDa)was purchased from Fermentas INC. Mouse Granzyme B and chloroquinediphosphate were purchased from Sigma-Aldrich. Alamarblue (100 ml) waspurchased from Invitrogen.

Synthesis of HA-DA Conjugates.

HA (5 g, 12.5 mmol) was dissolved in 500 ml of distilled water. To thisdiethoxyethyl amine (DA) with different amounts (1.19 g, 8.93 mmol) or(2.38 g, 17.8 mmol) was added followed by NHS (1.16 g, 10.0 mmol) andEDC (2.40 g, 12.5 mmol) to initiate the conjugation reaction. As thereaction proceeded, the pH of the mixture was maintained at 4.7 by theaddition of 0.1M NaOH. The reaction mixture was stirred overnight atroom temperature and then the pH was adjusted to 7.0. The solution wastransferred to dialysis tubes with molecular cut-off of 1000 Da. Thetubes were dialyzed against 100 mM sodium chloride solution for 2 days,a mixture of distilled water and ethanol (3:1) for 1 day and distilledwater for 1 day, successively. The purified solution was lyophilized toobtain the HA-DA. The degree of substitution (the number of DA moleculesper 100 repeating units of HA) was calculated from ¹H NMR measurement bycomparing the ratio of the relative peak integrations of methyl protonsof DA and the methyl protons of HA. The degree of substitution was16.8%.

Synthesis of HA-EGCG Conjugates.

HA-DA conjugates (1 g) were dissolved in 57 ml of distilled water. Thesolution was then degassed by bubbling nitrogen through the solution for20 min. EGCG solution was dissolved in 13 ml of degassed DMSO. The EGCGsolution (20 equivalents of molar concentration with respect to the DAunits) was then added to the solution of HA-DA conjugate. The pH of thesolution was adjusted to 1.0 using concentrated HCl. The resultingsolution was then stirred at room temperature for 24 h under a nitrogenatmosphere. The solution was transferred to dialysis tubes with amolecular cut-off of 3500 Da and dialyzed against water under a nitrogenatmosphere for 3 days. The purified solution was lyophilized to obtainthe HA-EGCG conjugate. The degree of substitution (the number ofbis-EGCG molecules per 100 repeating units of HA) was calculated by theUV-VIS measurements.

Synthesis of HA-EGCG-Rhodamine (HAER).

100 mg of HA-EGCG was dissolved in 10 ml of distilled water that hadbeen continuously degassed with nitrogen for 15 min. 1 mg of Lissaminerhodamine B ethyldiamine (Invitrogen) was dissolved in 1 ml of DMSO,protected from light, and degassed with nitrogen for 15 min. Therhodamine B solution was then added to HA-EGCG solution and the pH wasadjusted rapidly to 4.5 to facilitate the Schiff base formation betweenthe primary amine on Lissamine rhodamine B ethyldiamine and the freealdehyde groups on the main chain of HA-EGCG. Meanwhile, continuouspurging of the reaction mixture with nitrogen was carried out. The pHwas maintained for 3 h, after which the reaction mixture was dialyzedagainst 100 mM sodium chloride solution for 2 days, a mixture ofdistilled water and ethanol (3:1) for 1 day and distilled water for 1day, successively and always under a nitrogen atmosphere. Thefluorescence of the final dialysate was measured to determine whetherfree rhodamine dye (EX: 560-575 nm, EM: 580-595 nm) was still present.If so, dialysis was carried out for one more day in distilled water. Thepurified solution was lyophilized to obtain the HA-EGCG-rhodamine.

Synthesis of FITC-Lysozyme (FL).

300 mg of lysozyme was dissolved in 60 ml of 0.1 M borate buffer (pH8.5). 12 mg of FITC was dissolved in another 60 ml of borate buffer. TheFITC solution was added drop-wise to the lysozyme solution whilestirring in the dark. The solution was left stirring gently for 16 h,after which it was dialyzed against distilled water at 4° C. for 3 days,refreshing the distilled water twice daily. The resultant solution waslyophilized.

Preparation of HA-EGCG/Lysozyme Particles.

HA-EGCG was dissolved in distilled water at a stock concentration of 10mg/ml by vortexing for 5 min and sonication for 10 min. This HA-EGCGstock solution was subsequently diluted in PBS to the respective workingconcentrations. Lysozyme was dissolved in PBS. Particles weresynthesized at room temperature by simply mixing the HA-EGCG andlysozyme solutions with gentle pipetting, and then leaving the mixturesto sit for 10 min. The composition of the particles was varied bychanging the working concentrations of either HA-EGCG or lysozyme.

Dynamic Light Scattering (DLS).

The size of HA-EGCG/lysozyme particle was evaluated by DLS using aparticle sizer (Brookhaven instruments Co.) The DLS measurement wascarried out at a concentration of 0.75, 1 or 2 mg/ml samples at 25° C.

Fluorescence Quenching Studies.

The fluorescence emission spectra of the HA-EGCG/lysozyme particles,HA/lysozyme mixtures, free lysozyme alone, free HA-EGCG carrier or freeHA alone were measured at an excitation wavelength of 280 nm using afluorescence spectrophotometer (Hitachi, Japan). Difference spectrabetween the HA-EGCG/lysozyme particles and HA-EGCG were obtained todetermine the net fluorescence of lysozyme in particles of varyingHA-EGCG concentrations. The same was done for HA/lysozyme mixtures.

Lysozyme Activity Assay.

To determine the activity of lysozyme, 20 μl of sample containing eitherfree lysozyme or HA-EGCG/lysozyme particles was added to a well of a96-well assay plate, followed by 100 μl of M. lysodeikticus (0.15% (w/v)in PBS). The decrease in turbidity was monitored by a Tecan Infinite 200microplate reader by measuring the absorbance of the sample at 450 nm atroom temperature every 3 min for 15 min. The absorbance decay plot wasfitted to a linear equation, and the slopes were used to determineiysozyme activity. Regarding the restoration of activity of lysozymethat is complexed with HA-EGCG, the following protocol was used: theHA-EGCG/lysozyme particles were first allowed to self-assemble for 10min at room temperature, with free lysozyme diluted with PBS as acontrol. A constant volume of Triton-X of different concentrations wasthen added to each sample (to a final concentration of 0.001, 0.01 or0.1% w/v), incubated at room temperature for a further 5 min before thelysozyme substrate, M. lysodeikticus, was added, and the absorbancemonitored as described above.

Circular Dichroism (CD) Spectroscopy.

Far-ultraviolet CD spectra were obtained using an Olisspectropolarimeter. A cylindrical quartz cuvette with 0.5 mm path lengthwas used. The ellipticity was measured every 1 nm with an integrationtime of 1 s. Three sequential spectra were recorded and averaged foreach sample. The difference spectrum between the HA-EGCG/lysozymeparticles and HA-EGCG carrier was obtained and compared with thespectrum of lysozyme alone. The results are expressed in terms ofmillidegrees.

Cell Culture.

HCT-116 human colon carcinoma cells were obtained from ATCC, andcultured in McCoy's 5A medium supplemented with 10% FBS and 100 units/mLof penicillin/streptomycin.

Cellular Uptake Studies.

HCT-116 cells were seeded at 20,000 cells per well in 8-well LabTekchambered glass slides with complete growth medium and left to attachfor 48 h. The medium was aspirated and the cells were gently washed withserum-free medium once before FL, HAER, HAR, HAER/FL, HAR/FL, (alldiluted in serum-free medium) were added and the cells were incubated atgrowth conditions for 4 h respectively. The solutions were then removed,and the cells were washed twice with PBS, fixed with 4% formaldehyde atroom temperature for 15 min, washed twice with PBS, stained with 1 μg/mlof Hoechst 33358 to elucidate the nuclei, and finally washed twice withPBS. Confocal images were obtained with a Carl Zeiss LSM 5 DUO invertedconfocal microscope with a 63× objective and 488 nm, 543 nm lasers forexcitation of FITC and rhodamine B respectively. The confocal imageswere quantified with Metamorph program. The background fluorescenceintensity of 25 random spots was measured, averaged, and subtracted fromthe average FITC or rhodamine B fluorescence intensity of 25 randomspots that represented the extent of lysozyme and HAER/HAR uptake,respectively.

Cell Viability Studies:

HCT-116 cells were seeded at 10,000 cells per well in 96-wellmicroplates with complete growth medium and left to attach for 48 h.Test reagents were prepared by first mixing the linear PEI(polyethylenimine) or chloroquine solution (fixed at 100 μM)) withGranzyme B (fixed at 2 μg/ml) for 15 min before adding HA-EGCG solutionto the mixture and allow complexation to take place for 45 min. Themedium in the wells was then aspirated and the cells were treated withthe respective reagents for 48 h (all dilutions were made in serum-freemedium). Cell viability was analyzed using AlamarBlue. Before analysis,the medium was aspirated and the cells were gently washed with PBS oncebefore a mixture of 10 μl of alamarblue reagent with 100 μl ofserum-containing medium was added to each well. The cells were incubatedfor 2 h and the fluorescence emission of each well was measured using aTecan infinite Microplate Reader. The viabilities of treated cells wereevaluated as a percentage of the negative control. Each data set is anaverage of 4 repeats.

Results

HA-EGCG was synthesized by a two-step reaction procedure, as previouslydescribed. HA-diethoxyethylamine (HA-DA) conjugate was first prepared bya standard carbodiimide/active ester mediated-coupling reaction. Thiswas followed by the conjugation of EGCG to HA via a Baeyeracid-catalyzed reaction between a nucleophilic A ring of EGCG and analdehyde group which was formed by the deprotection of the diethoxyacetal group of HA-DA under acidic conditions (FIG. 2).

The complexation behavior between HA-EGCG and a model protein, lysozyme,was first studied by dynamic light scattering. Particles of HA-EGCG andlysozyme that were on the order of 100 nm could be prepared.HA-EGCG/lysozyme particles prepared with a fixed concentration ofHA-EGCG and varying lysozyme concentrations showed a biphasic behavior,with an initial decrease in particle size and a subsequent increase inparticle size with increasing lysozyme concentration (FIG. 3). As acontrol, a fixed concentration of unmodified HA was mixed with lysozymeof different concentrations, and it was observed that there was amonotonic decrease in particle size with increasing lysozymeconcentrations. This difference may be attributed to the hydrophobicEGCG moiety, which may allow the HA-EGCG particles to be formed via bothionic and hydrophobic interactions. In fact, at very high lysozymeconcentrations (>0.75% w/v), HA-EGCG/lysozyme particles becomeincreasingly turbid and start to phase separate from solution when leftto stand due to extensive hydrophobic interactions. In contrast,HA/lysozyme mixtures do not phase separate at the same proteinconcentrations.

The sample quality of HA-EGCG/lysozyme particles was also consistentlyhigher than that of HA/lysozyme mixtures (FIG. 4). Sample quality refersto the difference between the measured baseline and calculated baselineof the autocorrelation function, and in practical terms, a low samplequality indicates the presence of large particles or aggregates. Hence,HA-EGCG/lysozyme particles are more stable and homogeneous thanHA/lysozyme mixtures.

To confirm that the EGCG moiety is indeed necessary for HA-EGCG toefficiently bind protein, use was made of the fact that EGCG can quenchthe intrinsic fluorescence of lysozyme¹³ in a concentration-dependentmanner (FIG. 5). It was observed that HA-EGCG significantly decreasesintrinsic lysozyme fluorescence compared with HA (FIGS. 6 a & 6 b).Furthermore, it was noted that free EGCG quenches intrinsic lysozymefluorescence to a smaller degree than HA-EGCG with the same EGCGconcentration (FIG. 7), which shows that the conjugation of EGCG to HAenhances the binding interaction between EGCG and lysozyme, possiblybecause the HA backbone increases the local concentration of EGCGmoieties around a lysozyme molecule.

Deducing that the quenching of intrinsic lysozyme fluorescence byHA-EGCG could correlate to a partial denaturation of lysozyme, thesecondary structure of lysozyme was studied in its free form and inHA-EGCG/lysozyme particles by circular dichroism spectroscopy. Thedifference spectrum of HA-EGCG/lysozyme and HA-EGCG alone was obtainedto extract information about the lysozyme secondary structure. Theellipticity at 220 nm is a standard measure of helical content of aprotein and was used to estimate the secondary structural change of theprotein. As HA-EGCG concentration increases, the ellipticity at 220 nmdecreased (FIG. 8), indicating a partial denaturation of lysozyme.

The complexation of HA-EGCG with lysozyme can also be shown by aninhibition in lysozyme activity with an increase in HA-EGCGconcentration. Using M. lysodeikticus as the lysozyme substrate¹⁴, itwas found that HA-EGCG decreased lysozyme activity in aconcentration-dependent manner, whereas HA does not (FIG. 9 a).

As delivery of a functional protein into the cell was sought, efficientcomplexation was not sufficient. Dissociation of the complex to releasethe functional protein was also desired once the particle isinternalized within the cell, so that the protein can achieve itsintended function. In order to demonstrate that this is possible, thepre-formed HA-EGCG/lysozyme particles were destablized with Triton-X,which is expected to disrupt the hydrophobic interactions¹⁵ within thecomplex. For the free lysozyme control, it was noted that merely addinga low concentration of Triton-X (0.001% w/v) increases its apparentactivity to 140% of its original activity (FIG. 9 b). This isreasonable, considering that some free lysozyme can adhere to thehydrophobic plastic well and become partially denatured in the absenceof Triton-X. Further increasing Triton-X concentrations by two orders ofmagnitude (0.01% and 0.1%) result in a plateau in the apparent lysozymeactivity, which reaches its maximum activity at about 160%. For theHA-EGCG/lysozyme (comprising 50 μM HA-EGCG and 20 μg/ml lysozyme)complexes, adding 0.001% or 0.01% Triton-X did not significantly restorelysozyme activity, which remained close to 60% (FIG. 9 b). However, when0.1% Triton-X was added, sufficient disruption of hydrophobicinteractions within the HA-EGCG/lysozyme particles occurred, resultingin an almost complete restoration of lysozyme activity to its maximumlevel under these experimental conditions.

It was found that rhodamine B-labelled HA-EGCG (HAER) could beinternalized by HCT-116 human colon carcinoma cells more efficientlythan rhodamine B-labelled HA (HAR) (FIG. 10 a). Furthermore, particlesof unlabelled HA-EGCG and FITC-labelled lysozyme (HAE/FL) were found tomore efficiently deliver lysozyme into HCT-116 cells than eithermixtures of HA and FITC-labelled lysozyme (HA/FL) or without a deliverysystem (free protein, FL, itself) (FIG. 10 b). This is reasonable, sinceHA/lysozyme mixtures do not form very good complexes, and free lysozymeis like taken up by pinocytosis, which is much less efficient thatreceptor-mediated endocytosis. The colocalization of rhodamine B andFITC in cells treated with HAER/FL particles further confirms that theincreased uptake of lysozyme is indeed due to HA-EGCG acting as acarrier for lysozyme (FIG. 10 c).

In order to further develop the concept of intracellular delivery of afunctional protein using the present HA-EGCG particles, Granzyme B wasused. Granzyme B is a protease normally secreted by the Cytotoxic TLymphocytes (CTLs) of the immune system to kill virus-infected cells. Itdoes so through the activation of caspase intracellularly which triggersa cascade of reactions that induce apoptosis. Granzyme, when deliveredtogether with chloroquine, induced cytotoxicity in HCT-116 cells (datanot shown). Chloroquine is an endosomolytic agent that can facilitatethe escape of cargoes from endosomes. It is necessary to combinechloroquine with Granzyme B when treating the cells to allow Granzyme Bto be released into the cytosol where it can activate its target. Also,particles comprising HA-EGCG and Granzyme in the presence of chloroquineshowed apoptosis of the cell. However, the particles containingchloroquine are not appropriate due to the toxicity of chloroquine.Therefore, particles were designed comprising HA-EGCG, Granzyme and PEIfor intracellular delivery of protein drugs. It is well known that PEIis an endosomolytic agent and destabilizes endosomes in cells. It wasobserved that the cell viability decreased by using particles containingPEI (FIG. 11). 5 μg/ml of HA-EGCG showed the most significant Granzymeeffect. Also, the effect of Granzyme decreased when higher concentrationof HA-EGCG was utilized. These results indicate that the combination ofthe processes of destabilization of HA-EGCG and Granzyme complex andescape of Granzyme from endosomes is possibly most efficient when 5mg/ml HA-EGCG was utilized. From the results, it was concluded that theHA-EGCG particles have achieved successful delivery of a functionalintrabody, Granzyme B, into the cytosol of HCT-116 cells.

Although various embodiments of the invention are disclosed herein, manyadaptations and modifications may be made within the scope of theinvention in accordance with the common general knowledge of thoseskilled in this art. Such modifications include the substitution ofknown equivalents for any aspect of the invention in order to achievethe same result in substantially the same way.

Numeric ranges are inclusive of the numbers defining the range.

All technical and scientific terms used herein have the same meaning ascommonly understood by one of ordinary skill in the art of thisinvention, unless defined otherwise.

Concentrations given in this specification, when given in terms ofpercentages, include weight/weight (w/w), weight/volume (w/v) andvolume/volume (v/v) percentages.

The word “comprising” is used herein as an open-ended term,substantially equivalent to the phrase “including, but not limited to”,and the word “comprises” has a corresponding meaning. As used herein,the singular forms “a”, “an” and “the” include plural referents unlessthe context clearly dictates otherwise. Thus, for example, reference to“a thing” includes more than one such thing.

Citation of references herein is not an admission that such referencesare prior art to the present invention. Any priority document(s) and allpublications, including but not limited to patents and patentapplications, cited in this specification are incorporated herein byreference as if each individual publication were specifically andindividually indicated to be incorporated by reference herein and asthough fully set forth herein.

The invention includes all embodiments and variations substantially ashereinbefore described and with reference to the examples and drawings.In some embodiments, the invention excludes steps that involve medicalor surgical treatment.

REFERENCES

-   1. Park, J. W.; Kirpotin, D. B.; Hong, K.; Shalaby. R.; Shao, Y.;    Nielsen, U. B.: Marks, J. D.; Papahadjopoulos. D.; Benz. C. C.,    Tumor targeting using anti-her2 immunoliposomes. J Control Release    2001, 74, (1-3), 95-113.-   2. Senter, P. D.; Springer, C. J., Selective activation of    anticancer prodrugs by monoclonal antibody-enzyme conjugates. Adv    Drug Deliv Rev 2001, 53, (3), 247-64.-   3. Wheeler, Y. Y.; Chen, S. Y.; Sane, D. C., Intrabody and intrakine    strategies for molecular therapy. Mol Ther 2003, 8, (3), 355-66.-   4. Kontermann, R. E., Intrabodies as therapeutic agents. Methods    2004, 34, (2), 163-70.-   5. Tse, E.; Rabbitts, T. H., Intracellular antibody-caspase-mediated    cell killing: an approach for application in cancer therapy. Proc    Natl Acad USA 2000, 97, (22), 12266-71.-   6. Heng, B. C.; Cao, T., Making cell-permeable antibodies    (Transbody) through fusion of protein transduction domains (PTD)    with single chain variable fragment (scFv) antibodies: potential    advantages over antibodies expressed within the intracellular    environment (Intrabody). Med Hypotheses 2005, 64, (6), 1105-8.-   7. Bourguignon, L. Y.; Zhu, H.; Shao, L.; Chen, Y. W., CD44    interaction with tiamI promotes RacI signaling and hyaluronic    acid-mediated breast tumor cell migration. J Biol Chem 2000, 275,    (3), 1829-38.-   8. Matsubara, Y.; Katoh, S.; Taniguchii, H.; Oka, M.; Kadota, J.;    Kohno, S., Expression of CD44 variants in lung cancer and its    relationship to hyaluronan binding. J Int Med Res 2000, 28, (2),    78-90.-   9. Shimizu, T.; Kishida T.; Hasegawa, U.; Ueda, Y.; Imanishi, J.;    Yamagishi, H.; Akiyoshi, K.; Otsuji, E.; Mazda, O., Nanogel DDS    enables sustained release of IL-12 for tumor immunotherapy. Biochem    Biophys Res Commun 2008, 367, (2), 330-5.-   10. Kirker, K. R.; Luo, Y.; Nielson, J. H.; Shelby, J.;    Prestwich, G. D., Glycosaminoglycan hydrogel films as    bio-interactive dressings for wound healing. Biomaterials 2002, 23,    (17), 3661-71.-   11. Maiti, T. K.; Ghosh, K. S.; Dasgupta, S., Interaction of    (−)-epigallocatechin-3-gallate with human serum albumin:    fluorescence, fourier transform infrared, circular dichroism, and    docking studies. Proteins 2006, 64, (2), 355-62.-   12. Abe. I.; Kashiwagi, K.; Noguchi, H., Antioxidative galloyl    esters as enzyme inhibitors of p-hydroxybenzoate hydroxylase. FEBS    Lett 2000, 483, (2-3), 131-4.-   13. Rawel, H. M., Frey, S. K.; Meidtner, K.; Kroll, J.;    Schweigert. F. J., Determining the binding affinities of phenolic    compounds to proteins by quenching of the intrinsic tryptophan    fluorescence. Mol Nutr Food Res 2006, 50, (8), 705-13.-   14. Kurschus, F. C.; Kleinschmidt. M.; Fellows, E.; Dornmair, K.;    Rudolph, R.; Lille, H.; Jenne, D. E., Killing of target cells by    redirected granzyme B in the absence of perforin. FEBS Lett 2004,    562, (1-3), 87-92.-   15. Zordan-Nudo, T.; Ling, V.; Liu, L.; Georges, E., Effects of    nonionic detergents on P-glycoprotein drug binding and reversal of    multidrug resistance. Cancer Res 1993, 53, (24), 5994-6000.-   16. Jankun J., et al. Nature 387, 561 (1997).-   17. Bodoni A. et al. J. Nutr. Biochem. 13, 103-111 (2002).-   18. Nakagawa K. et al. J. Agric. Food Chem. 47, 3967-3973 (1999).-   19. Terao J., et al. Arch. Biochem. Biophys. 308, 278-284 (1994).-   20. Isemura M., et al. Biofactors 13, 81-85 (2000).-   21. Ikeda I., et al. J. Nutr. 135, 155 (2005).-   22. Lill G., et al. FEBS Letters 546, 265-270 (2003).-   23. Sakanaka S, and Okada Y. J. Agric. Food Chem. 52, 1688-1692    (2004).-   24. Yokozawa T., et al., J. Agric. Food Chem. 48, 5068-5073 (2000).-   25. Jankun, J., Selman, S. H., Swiercz, R., Why drinking green tea    could prevent cancer, Nature, 387, 561 (1997).-   26. Garbisa, S., Biggin, S., Cavallarin, N., Sartor, L., Benelli,    R., Albini, A., Tumor invasion: molecular shears blunted by green    tea, Nature medicine, 5(11), 1216 (1999).-   27. Tachibana, H., Koga, K., Fujimura, Y., Yamada, K., A receptor    for green tea polyphenol EGCG, Nature Structural & Molecular    Biology, 11(4), 380-381 (2004).-   28. Nagle, D. G., Ferreira, D., Zhou, Y. D.,    Epigallocatechin-3-gallate (EGCG): chemical and biomedical    perspectives, Phytochemistry, 67, 1849-1855 (2006).-   29. Sang, S., Yang, I., Buckley, B., Ho, C. T., Yang, C. S.,    Autooxidative quinine formation in vitro and metabolite formation in    vivo from tea polyphenol (−)-epigallocatechin-3-gallate: studied by    real-time mass spectrometry combined with tandem mass ion mapping,    Free Radical Biology & Medicine, 43, 362-371 (2007).-   30. Yang, C. S., Wang, Z. Y., Tea and cancer, Journal of the    National Cancer Institute, 85(13), 1038-1049 (1993).-   31. Garbisa, S., Sartor, L, Biggin, S., Salvato, B., Benelli, R.,    Albini, A., Tumor gelatinases and invasion inhibited by the green    tea flavanol epigallocatecin-3-gallate, Cancer, 91(4), 822-832,    (2001).

What is claimed is:
 1. A method for delivery of a bioactive agent to acell, the method comprising contacting the cell with a suspension ofimmiscible particles in an aqueous solution, wherein the particlescomprising an agglomeration of: a bioactive agent; and a plurality ofconjugates of a hyaluronic acid and a flavonoid; the particles having ahydrated interior and having an average diameter of from about 15 nm toabout 300 nm, the bioactive agent being releasably retained in theparticles by the flavonoid.
 2. The method of claim 1, wherein thebioactive agent is delivered intracellularly.
 3. The method of claim 1wherein the cell is a cancer cell.
 4. The method of 1 wherein the cellis in vitro.
 5. The method of claim 1 wherein the cell is in vivo, themethod further comprising administering the suspension to a subject inan amount effective for the treatment of cancer.
 6. A method offormulating a suspension of immiscible particles comprising combining inan aqueous solution: a. 0.01% w/v to about 1.0% w/v of a bioactiveagent; and b. 0.05 μg/ml to about 5000 μg/ml of conjugates of ahyaluronic acid and a flavonoid; to form the suspension of particles theparticles comprising an agglomeration of the bioactive agent and theconjugates of hyaluronic acid and a flavonoid, the particles having ahydrated interior and having an average diameter of from about 15 nm toabout 300 nm, the bioactive agent being releasably retained in theparticles by the flavonoid.
 7. The method of claim 6 comprisingcombining an endosomolytic agent with the bioactive agent and theconjugates of a hyaluronic acid and a flavonoid to form the suspensionand wherein the particles further comprise the endosomolytic agent. 8.The method of claim 6 further comprising formulating the suspension intoa therapeutic formulation.
 9. The method of claim 1 wherein theflavonoid is a catechin-based flavonoid.
 10. The method of claim 9wherein the flavonoid is epigallocatechin gallate.
 11. The method ofclaim 1 wherein the bioactive agent is an anti-cancer agent.
 12. Themethod of claim 1 wherein the bioactive agent is a protein.
 13. Themethod of claim 1 wherein the bioactive agent is an intrabody.
 14. Themethod of claim 1 wherein the bioactive agent is Granzyme B.
 15. Themethod of claim 1 wherein the particles further comprise anendosomolytic agent.
 16. The method of claim 6 wherein the bioactiveagent is releasably retained in the particles by a hydrophobic bond oran ionic bond between the flavonoid and the bioactive agent.
 17. Themethod of claim 6 wherein the particles are on average from about 50 nmto about 100 nm in diameter.
 18. The method of claim 6 wherein thehyaluronic acid has a molecular weight of from about 5000 to about10000000 daltons.